22-adrenergic receptor (22AR) signaling behavior is complex. The receptor can couple to G proteins through multiple signaling pathways in the same cell or signal without G protein. 22AR ligands have different efficacies or biases in different signaling pathways, sometimes leading to opposite activities. Moreover, the ability to rapidly regulate 22AR expression without stimulating traditional cell responses is of considerable pharmacological interest. Currently, generalized approaches for small molecule discovery for non-canonical ligands of GPCRs as well as ligands for orphan receptors are relatively unavailable to the research community. Here we propose the first homogeneous HTS discovery opportunities based on protein transport to and from the plasma membrane that requires neither microscopy nor antibody-based detection. The Carnegie Mellon University Technology Center for Networks and Pathway (CMU TCNP) has developed a novel fluorogen activating peptide (FAP) technology for GPCR activation and internalization and applied it first to the human 22AR. The University Of New Mexico Center for Molecular Discovery (UNMCMD, an NIH Roadmap Specialty Probe Production Center U54MH084690) has developed flow cytometric HTS multiplex technology, which allows multiple targets to be screened simultaneously. The proposed collaboration sets the stage for probe development for multiplex target families. In this project we will! 1. Perform HTS on the 22AR cells to identify molecules that result in internalization. We anticipate finding both canonical and non-canonical ligands that induce or prevent receptor internalization. 2. Use standard and novel secondary bead-based assays to classify molecules that induce internalization as: traditional ligands (agonists or antagonists) with potential pathway bias; non-canonical ligands which act selectively on individual receptors or generically on receptor pathways to cause internalization without receptor activation, and allosteric ligands which alter the responses of receptors to canonical ligands. Meeting these aims will promote the development and application of automated highly parallel screens that (1) identify non-canonical ligands for liganded GPCRs as well as novel ligands for orphan GPCRs, and (2) identify new receptor-regulatory compounds in a format that reveals responses by multiple GPCRs, not just one or a few. The highest value targets for this application will be those which induce internalization without activation. PUBLIC HEALTH RELEVANCE: This project will apply a new assay to identify canonical and noncanonical ligands that promote internalization of the beta-2adrenergic receptor. Because this and other GPCRs are among the most important targets of prescription drugs, the new assay has the potential to lead to a new generation of important therapeutic molecules.